Systems and methods for calibrating and correcting a speckle contrast flowmeter

ABSTRACT

Disclosed herein are systems, methods, and devices for calibrating contrast measurements from laser speckle imaging systems to accurately determine unknown particle motion characteristics, such as flow rate. The calibration stores to memory calibration data, which may include a set of measurements from samples with known particle characteristics and/or estimates of noise, including the effects on contrast arising from undesired signals unrelated to the unknown particle motion characteristics. The calibration data may be accessed and used to correct an empirical measurement of contrast and/or interpolate a value of the unknown particle motion characteristic. The system may include a light source, photodetector, processor, and memory, which can be combined into a single device, such as a wearable device, for providing calibrated flow measurements. The device may be used, for example, to measure blood flow, cardiac output, and heart rate, and can be used to amplify the pulsatile signal.

INCORPORATION BY REFERENCE

This application is a divisional application of U.S. patent applicationSer. No. 16/093,597, filed on Oct. 12, 2018, which is a national stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2017/028178, filed on Apr. 18, 2017, which claims priority benefitof U.S. Provisional Patent Application No. 62/324,903, filed Apr. 20,2016, and all of application Ser. Nos. 16/093,597, PCT/US2017/028178,and 62/324,903 are incorporated herein by reference in their entirety.Any and all applications related thereto by way of priority thereto ortherefrom are hereby incorporated by reference in their entirety.

BACKGROUND

This disclosure relates to devices, systems and methods for calibratingand correcting flowmetry measurements made using laser speckle imaging(LSI). LSI is an optical technique for determining the rate of motionwithin a sample using interferometric information. LSI is typicallyperformed with a coherent illumination source and image sensor, wherelight interrogates a sample and randomly interferes on the image sensor,producing a signature “speckle” pattern. The pattern is then analyzed,in space and/or in time, to determine particle motion within the sample.

Dynamic Light Scattering (DLS) is a technique for determining particlesize and fluid flow rate that utilizes coherent illumination andinterference. The technology has been used in medical applications forsome time to measure blood perfusion [1]. In recent years, DLStechnologies have seen major innovation and are now performed in avariety of ways [2]. One DLS method, called laser speckle imaging (LSI),uses a coherent laser source to illuminate a sample of light scatteringparticles, and images the scattered light using a multi-pixel detector.Early iterations used multiple photodetectors [3, 4], but manyinstruments now use a silicon-based camera sensor [5]. The sensorrecords the so-called “speckle” pattern, produced by light interference,as the scattered coherent light recombines onto the defection element.If the scattering particles are in motion, the interference pattern willfluctuate over time. The detection element has a finite exposure time,and if the interference pattern fluctuates during the exposure, thespeckles will “blur,” or their light intensity will be averaged withinthe detection element pixels. Researchers have previously developed amethodology to quantify the amount of “blurring” during the exposure byanalyzing the amount of contrast between pixel intensity values in timeand/or space. One common way to quantify contrast is to calculate thestandard deviation of a local neighborhood of pixel intensities, oftennormalizing to the mean [6]. This parameter is typically referred to asthe “speckle contrast.” A reduction in speckle contrast indicates anincrease in flow and vice versa. The speckle contrast may alternativelybe calculated for multiple frames in time.

LSI is a useful technology in biomedical research to study blood flowwithin vascularized tissue [7]. Cells and other structures within theblood scatter the coherent light as they flow through the vasculature,and LSI can quantify this flow. Further developments have seen theinclusion of Monte Carlo simulation results and static scatteringcomponents in the LSI model [8]. However, a major disadvantage to LSI isthat it is highly susceptible to numerous sources of noise. Because LSIrelies on the standard deviation between pixels, noise from randomand/or system sources, such as shot noise or dark sensor noise, canaffect the speckle contrast and hence impact the quantification of flow.A myriad of other factors may affect the formulation of the specklepattern onto the sensor including: coherence length of the laser (whichvaries between lasers and manufacturers), numerical aperture of theoptical system, pixel size, wavelength, and ambient light, among others[9].

SUMMARY

LSI is an optical technique for determining the rate of motion within asample using interferometric information. LSI is typically performedwith a coherent illumination source and image sensor, where lightinterrogates a sample and randomly interferes on the image sensor,producing a signature “speckle” pattern. The pattern is then analyzed,in space and/or in time, to determine particle motion within the sample.Particularly, this disclosure relates to ways to correct errors in theoutput given by laser speckle contrast analysis. Errors may arise whenundesired signals affect the speckle contrast. Generally these signalsare unrelated to particle motion characteristics of the light scatteringparticles in the interrogated sample. The effect of the undesiredsignals on the speckle contrast value may be determined throughcalibration steps involving measurements of known samples (samples withknown particle characteristics), or the effect may be estimated fromknown characteristics of the sensor, source, or other conditions. Thecorrection may account for and reduce or eliminate the errors caused bynon-flow elements that affect speckle contrast such as, but not limitedto: sensor noise, source coherence (due to fluctuations in laser powersupply voltage), statistical variance (natural variation in specklepattern statistics), and ambient light. More specifically, in aparticular non-limiting case, this method may be used in a clinicalsetting to determine a more accurate flow rate of blood cells withinvascularized tissue by eliminating the effect of camera noise on thespeckle image. In a second non-limiting case, this method may be used toincrease the amplitude of the pulse waveform caused by the cardiaccycle, by removing the components of the signal that arise fromnon-pulsatile elements.

In some embodiments, a system for determining unknown particle motioncharacteristics in a sample of interest using a calibrated contrastmeasurement from a laser speckle imaging device is disclosed. The systemincludes a laser speckle imaging device configured for contrastanalysis, a computer-readable memory storing calibration data, and aprocessor operably coupled to the detector and to the computer-readablememory. The laser speckle imaging device includes a light sourceconfigured to emit light such that the light scatters within a sampleand a photo-sensitive detector having one or more light-sensitive pixelelements configured to receive at least some of the scattered light. Thestored calibration data comprises one or more measurements of lightscattered from a calibration sample comprising light scatteringparticles with particle characteristics known a priori and data relatedto the known particle characteristics of the calibration sample and/ordata derived from the combined analysis of the measurements and data.The processor is programmed to derive a contrast measurement bycomparing light detected by the one or more pixels in time and/or spacethat has scattered from the sample of interest comprising lightscattering particles with unknown particle motion characteristics. Theprocessor is further programmed to read the stored calibration data fromthe computer-readable memory and calibrate the contrast measurement fromthe sample of interest by correlating the contrast measurement to thecalibration data so as to determine the unknown particle motioncharacteristics of the sample of interest.

Correlating the contrast measurement to the calibration data maycomprise evaluating a calibration function estimated from the one ormore measurements from the calibration sample. Correlating the contrastmeasurement to the calibration data may comprise interpolation orextrapolation of the one or more measurements from the calibrationsample. Correlating the contrast measurement to the calibration data maycomprise at least partially correcting the contrast measurement toaccount for a measure of noise arising from undesired signals, themeasure of noise being derived from the one or more measurements fromthe calibration sample. At least partially correcting the contrastmeasurement can comprise subtracting from the contrast measurement themeasure of noise or dividing the contrast measurement by the measure ofnoise. The measure of noise may account for one or more of detectornoise, light source coherence, statistical variance, and ambient orbackground light. The processor may be further programmed to store tothe computer-readable memory a calibration result made from determiningthe unknown particle motion characteristics, to read the storedcalibration result, and to calibrate subsequent measurements based onthe stored calibration result.

The light scattering particles of the sample of interest may be bloodcells and the unknown particle characteristics may be a measure of theflow rate of the blood cells. The one or more measurements from thecalibration sample may be acquired from the same laser speckle imagingdevice used to detect the light scattered from the sample of interest inderiving the contrast measurement. The one or more measurements from thecalibration sample may be acquired from a laser speckle imaging devicedistinct from that used to detect the light scattered from the sample ofinterest in deriving the contrast measurement. The one or moremeasurements from the calibration sample may include a measurement takenusing incoherent light. Correlating the contrast measurement to thecalibration data may comprise correcting the contrast measurement to beapproximately zero for the measurement taken using incoherent light. Thecalibration data may include a look-up table comprising pairs ofcontrast measurements from the calibration sample and known flow ratesof the light scattering particles of the calibration sample.

The laser speckle imaging device, the computer-readable memory, and theprocessor may be housed within a single device. The single device may beconfigured to be worn by a user to measure a sample of interest with inthe user. The laser speckle imaging device may be configured to measurepulsatile blood flow deriving from the cardiac cycle. The system mayinclude the calibration sample. The calibration sample may be a fluidcomprising light scattering particles, wherein the fluid is configuredto be pumped at known volumetric flow rates.

In some embodiments, a system for determining unknown particle motioncharacteristics in a sample of interest using a calibrated contrastmeasurement from a laser speckle imaging device is disclosed. The systemincludes a laser speckle imaging device configured for contrastanalysis, a computer-readable memory storing calibration data, and aprocessor operably coupled to the detector and to the computer-readablememory. The laser speckle imaging device includes a light sourceconfigured to emit light such that the light scatters within a sampleand a photo-sensitive detector having one or more light-sensitive pixelelements configured to receive at least some of the light. Thecalibration data includes an a priori estimate of the effect on contrastarising from signals unrelated to particle motion characteristics of thelight scattering particles in the sample of interest. The processor isprogrammed to derive an empirical measure of the total contrast in lightdetected by the one or more pixel elements in time and/or space that hasscattered from the sample of interest comprising light scatteringparticles with unknown particle motion characteristics. The processor isfurther programmed to calibrate the empirical measure of total contrastby using the a priori estimate to correct for contrast elements that areunrelated to particle motion characteristics of the light scatteringparticles of the sample of interest and determine the unknown particlemotion characteristics of the sample of interest from the calibratedempirical measure of total contrast.

The a priori estimate may be based on at least one previously recordedmeasurement. The at least one previously recorded measurement may havebeen taken using incoherent light. The at least one previously recordedmeasurement may have been recorded using the same laser speckle imagingdevice used to detect the light scattered from the sample of interest inderiving the empirical measure of the total contrast. The a prioriestimate may be based at least in part on the noise characteristics ofthe detector. The a priori estimate may be based at least in part onambient or background light. The a priori estimate may be based at leastin part on light intensity variation not due to interference. The lightscattering particles of the sample of interest may be blood cells andthe unknown particle characteristics may include a measure of the flowrate of the blood cells. The empirical measure of total contrast may bea measure of pixel variance and the a priori estimate may be a measureof pixel variance. Correcting the empirical measure of total contrastmay comprise subtracting or ratioing the a priori estimate of variancefrom the empirical measure of variance.

The laser speckle imaging device, the computer-readable memory, and theprocessor may be housed within a single device. The single device may beconfigured to be worn by a user to measure a sample of interest withinthe user. The laser speckle imaging device may be configured to measurepulsatile blood flow deriving from the cardiac cycle.

In some embodiments, a method for determining unknown particle motioncharacteristics in a sample of interest using a calibrated contrastmeasurement from a laser speckle imaging device is disclosed. The methodcomprises employing a laser speckle imaging device configured forcontrast analysis to obtain a measurement of light scattered from asample of interest comprising light scattering particles with unknownparticle motion characteristics. The laser speckle imaging deviceincludes a light source configured to emit light such that the lightscatters within a sample and a photo-sensitive detector having one ormore light-sensitive pixel elements configured to receive at least someof the scattered light. The method further comprises accessingcalibration data from a computer-readable memory. The calibration dataincludes one or more measurements of light scattered from a calibrationsample comprising light scattering particles with particlecharacteristics known a priori and data related to the known particlecharacteristics of the calibration sample and/or data derived from thecombined analysis of the one or more measurements and the data. Themethod further comprises deriving a contrast measurement by comparinglight detected by the one or more pixels in time and/or space from themeasurement of light and calibrating the contrast measurement from thesample of interest by correlating the contrast measurement to thecalibration data so as to determine the unknown particle motioncharacteristics of the sample of interest.

The method may further comprise employing the laser speckle imagingdevice to obtain the one or more measurements from the calibrationsample. The calibration sample may be a fluid comprising lightscattering particles with particle characteristics known a priori andthe method may further comprising pumping the fluid at a known flowrate. Pumping the fluid at a known flow rate may comprise pumping thefluid at two or more different known flow rates.

The calibration sample may be a living subject, and the method mayfurther comprise occluding blood flow within an extremity of the subjectto reduce or cause a cessation of blood flow. Occluding blood flow maycomprise applying a blood-pressure cuff to the ankle, legs, or arms ofthe subject.

The method may further comprise illuminating the calibration sample withincoherent light to obtain the one or more measurements from thecalibration sample. The method may further comprise storing a resultfrom the calibration to the computer readable memory; employing thelaser speckle imaging device to obtain a subsequent measurement of lightscattered from the same or a different sample of interest comprisinglight scattering particles with unknown particle motion characteristics;accessing the calibration result from the computer-readable memory;deriving a subsequent contrast measurement by comparing light detectedby the one or more pixels in time and/or space from the subsequentmeasurement of light; and calibrating the subsequent contrastmeasurement by correlating the subsequent contrast measurement to thecalibration result so as to determine the unknown particle motioncharacteristics. The light scattering particles of the sample ofinterest may be blood cells and determining the unknown particlecharacteristics may comprise determining the flow rate of the bloodcells.

In some embodiments, a method for determining unknown particle motioncharacteristics in a sample of interest using a calibrated contrastmeasurement from a laser speckle imaging device is disclosed. The methodmay comprise employing a laser speckle imaging device configured forcontrast analysis comprising to obtain a measurement of light scatteredfrom a sample of interest comprising light scattering particles withunknown particle motion characteristics. The laser speckle imagingdevice includes a light source configured to emit light such that thelight scatters within a sample and a photo-sensitive detector having oneor more light-sensitive pixel elements configured to receive at leastsome of the scattered light. The method further comprises accessing fromcomputer-readable memory an a priori estimate of the effect on contrastarising from signals unrelated to particle motion characteristics of thelight scattering particles of the sample of interest. The method furthercomprises deriving an empirical measure of the total contrast in lightdetected by the one or more pixel elements in time and/or space from themeasurement of light. The method further comprises calibrating theempirical measure of total contrast by using the a priori estimate tocorrect for contrast elements that are unrelated to particle motioncharacteristics of the light scattering particles of the sample ofinterest and determining the unknown particle motion characteristics ofthe sample of interest from the calibrated empirical measure of totalcontrast.

The method may further comprise employing the laser speckle imagingdevice to obtain the a priori estimate. Employing the laser speckleimaging device to obtain the a priori estimate may comprise pumpingfluid comprising light scattering particles with particlecharacteristics known a priori at a known flow rate and measuring lightscattered from the light scattering particles with particlecharacteristics known a priori. Pumping the fluid at a known flow ratemay comprise pumping the fluid at two or more different known flowrates. Employing the laser speckle imaging device to obtain the a prioriestimate may comprise occluding blood flow within an extremity of aliving subject to reduce or cause a cessation of blood flow andmeasuring light scattered from the occluded extremity of the subject.Occluding blood flow may comprise applying a blood-pressure cuff to theankle, legs, or arms of the subject.

The method may further comprise illuminating a calibration sample withincoherent light to obtain the a priori estimate. The method may furthercomprise employing the laser speckle imaging device to obtain asubsequent measurement of light scattered from the same or a differentsample of interest comprising light scattering particles with unknownparticle motion characteristics; accessing the a priori estimate fromthe computer-readable memory; deriving a subsequent empirical measure ofthe total contrast in light detected by the one or more pixel elementsin time and/or space from the subsequent measurement of light,calibrating the subsequent empirical measure of total contrast by usingthe a priori estimate to correct for contrast elements that areunrelated to particle motion characteristics of the light scatteringparticles; and determining the unknown particle motion characteristicsfrom the calibrated subsequent empirical measure of total contrast. Thelight scattering particles of the sample of interest maybe blood cellsand determining the unknown particle characteristics may comprisedetermining the flow rate of the blood cells.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentdisclosure will now be described with reference to the drawings ofembodiments, which embodiments are intended to illustrate and not tolimit the disclosure. One of ordinary skill in the art would readilyappreciate that the features depicted in the illustrative embodimentsare capable of combination in manners that are not explicitly depicted,but are both envisioned and disclosed herein.

FIGS. 1A-1D schematically illustrate various system configurations. FIG.1A shows the system in a reflectance, non-contact configuration. FIG. 1Bshows the system in a transmission, non-contact configuration. FIG. 1Cshows the system in a reflectance, contact configuration. FIG. 1D showsthe system in a transmission, contact configuration.

FIGS. 2A-2B illustrates an example of an interrogation device. FIG. 2Aschematically illustrates use of the interrogation device totransilluminate a subject's digit. FIG. 2B illustrates the interrogationdevice coupled to an external processor with display.

FIG. 3 schematically illustrates the components of an example systemincluding an interrogation device coupled to a computer comprising aprocessor and memory.

FIG. 4 illustrates an example of expected contrast error measured for aphotodetector illuminated by different intensities of incoherent light.

FIG. 5 illustrates an example of flow indices measured by LSI plottedagainst known values of the true flow rare for a calibration sample.

FIG. 6 illustrates a before-and-after example of using the disclosedcalibration to improve the photodetector output of an LSI system used tomeasure the pulsatile blood flow of a subject.

DETAILED DESCRIPTION

The systems, devices, and methods disclosed herein may incorporatecomponent devices, including a light source 100, a photodetector 200(i.e. a photosensitive detector, such as an image sensor), memory, andone or more processors, which may be operatively connected to oneanother to interrogate a sample 300. In many embodiments, the sample maybe a physiological sample, such as a region of tissue on a subject,about which physiological information is to be ascertained. The subjectmay be a living animal, such as a human. The component devices may bestandard devices employed in new configurations, methodologies, and/orsystems or they may be devices specifically designed or adapted toperform in the systems and methods disclosed herein. The light source100 may he configured to emit at least partially coherent light. Thelight source 100 may he a laser, such as a diode laser. In someembodiments, the light source 100 is a VCSEL laser. The photodetector200 may comprise one or more light-sensitive elements (e.g., pixels) fordetecting light recovered from the light source 100 after interactionwith a sample. The photodetector 200 may, for example, be asilicon-based camera sensor. The camera sensor may be of any suitabletype, including but not limited to CMOS or CCD image sensors. Thephotodetector 200 may be configured to generate one or more signalsrelated to the detected light and to transmit these signals to theprocessor. The signals may comprise quantifiable information about theintensity of light detected at one or more pixels at a point in time orover a course of time. In some embodiments, the signals may compriseinformation about the wavelength(s) of the detected light. The signalsmay be analog or digital. If the signals are analog they may besubsequently converted into digital signals either before or after beingtransmitted from the photodetector 200.

The light source 100 and photodetector 200 may be positionable in anynumber of configurations relative to the sample 300 including but notlimited to being placed in contact or noncontact geometries, or inreflectance or transmission geometries, as seen in FIGS. 1A-1D. Thedevices are positionable in that they can each be maintained in arelatively constant spatial orientation relative to the sample 300during the measurement so that changes in the detected signal resultingfrom movement of the light source 100, photodetector 200, and/or sample300 relative to one another are negligible relative to the informationalcontent attained from the sample 300. The positionable devices may beaffixed to each other, part of an integral device, or distinctstructures. One or both of the devices may be removably attached to thesample, such as affixed to a surface of the sample, or they may befree-standing or affixed to a structure independent of the sample 300.At least a portion of the light emitted from a positionable light source100 is able to reach a surface of the sample 300 and at least a portionof the light detected by a positionable photodetector 200 has contactedthe sample 300. FIG. 1A shows a non-contact reflectance geometry whereinthe light source 100 and photodetector 200 are both positioned on thesame side of the sample 300, neither of which is in direct physicalcontact with a surface of the sample 300. The photodetector 200 isconfigured to receive light reflected from the surface of the sample 300as well as light scattered internally within the sample. FIG. 1B shows anon-contact transmission geometry wherein the light source 100 and thephotodetector 200 are positioned on opposite sides of the sample 300through which the light emitted from the light source 100 passes throughand in which neither the light source 100 nor the photodetector 200 arein direct physical contact with a surface of the sample 300. The lightsource 100 and photodetector 200 may or may not be positioned directlyacross from each other in a transmission geometry. FIG. 1C shows acontact reflectance geometry wherein the light source 100 and thephotodetector 200 are both positioned on the same side of the sample300, both of which are in direct physical contact with a surface of thesample 300. FIG. 1D shows a contact transmission geometry wherein thelight source 100 and photodetector 200 are positioned on opposite sidesof the sample 300 through which the light emitted from the light source100 passes through and in which both the light source 100 and thephotodetector 200 are in direct physical contact with a surface of thesample 300. Variations are also possible for each geometry wherein oneof the light source 100 and the photodetector 200 is in direct physicalcontact with a surface of the sample 300 and the other is not. Thesegeometries as described and illustrated in FIGS. 1A-1D are non-limitingexamples and the systems and methods disclosed herein may be practicedwith any suitable configuration of the system components. For example,the photodetector 200 may be positioned in a configuration that neitherreceives surface-reflected light nor transmitted light.

In many embodiments, coherent light or at least partially coherent lightis emitted by the light source 100 and directed toward the sample 300.The photodetector 200 is positioned to recover at least some of thelight emitted by the light source 100 after it has interacted with thesample 300. In various embodiments, the device, system, or method may beconfigured to maximize collection of light scattered from lightscattering particles within the sample 300, particularly light scatteredfrom light scattering particles undergoing flow (e.g., blood cells) orother types of motion (e.g., diffusion). The light emitted by the lightsource 100 may be emitted at a constant intensity over a time sufficientfor detection. In other embodiments, the light may be emitted accordingto dynamic patterns. In many embodiments, the light may be emitted anddetected over a period of time sufficient to detect changes which occurin the sample 300 and which alter the path of the emitted light and/orproperties of the detected light. For example, by recording oversufficient time frames, dynamic properties of light scatteringparticles, such as a rate of motion (e.g., flow rate) can be observed.The processor may be used to record the signal(s) detected by thephotodetector 200 over time to memory and/or analyze the signals and/orthe temporal changes in the signals over time to determine informationabout the sample 300, such as unknown particle motion characteristics oflight scattering particles in the sample 300.

FIGS. 2A and 2B illustrate examples of an interrogation device 400,which is configured as a finger clip to interrogate blood flow withinvascularized tissue of a digit (e.g., finger). FIG. 2A schematicallyillustrates the transillumination of a portion of the finger coupled tothe interrogation device 400. FIG. 2B illustrates the interrogationdevice operatively coupled to an external processor 500. Theinterrogation device 400 can include the light source 100 andphotodetector 200 in an integrated or joinable housing, as shown inFIGS. 2A and 2B. The finger clip 400 may be configured to operate in anyconfiguration (e.g., transmission or reflectance as well as contact ornon-contact). Some embodiments of the interrogation device 400 may beconfigured to be wearable or attachable to a subject. These may include,but are not limited to, belts, wrist-bonds, skin patches, ear-clips,etc. The interrogation device 400 may be operatively coupled to theprocessor 500 by a data cable 402, which may transfer data and/or powerbetween the interrogation device 400 and the processor 500. The datacable 402 may be a USB cable or any other suitable cable. In someembodiments, the interrogation device 400 may include wirelessfunctionality for operatively coupling to the processor 500. Theprocessor 500 can include a display 502 for displaying data, such as adetected waveform, an image of a spectral pattern, a histogram of data,etc.

FIG. 3 schematically illustrates the interaction of the components of anexample interrogation device 400 and a computer. The processor 500 canbe part of a computer, a tablet, or any other suitable device. Thecomputer may further include a memory, a display, audio devices, and/orother components. The computer may comprise a PC USB hub for operativelycoupling to the interrogation device 400. In some embodiments, a display502 may be separate from the processor 500. In some embodiments, theinterrogation device 400 can include a display. The interrogation device400 can include the light source 100 (e.g., a laser diode) and/or thephotodetector 200. In the example shown in FIG. 3 , the light source 100and the photodetector 200 are configured in a transmission geometryaround a sample 300 of physiological tissue. The processor 500 mayreceive information from the photodetector 200, such as receivegenerated signals, and from the light source 100, and send instructionsfor controlling operation of the light source 100 and the photodetector200. In some embodiments, the systems may incorporate feedback formodulating the emission of light from the light source 100 and/or thedetection of light by the photodetector 200 according to an analysis ofthe detected light and/or generated signals by the processor 500.

The processor 500 may be operatively coupled to memory, which may becomprised of one or more memory components. The memory may be integralwith the processor (e.g., part of an integrated chip) and/or may beexternal to the processor 500. The processor 500 may be configured toread and/or write to memory. For example, the processor 500 may beconfigured to store raw input from the photodetector 200 to the memory(e.g., raw measurements of light intensity, time points, pixelidentifications) and/or may store processed or partially processed inputto the memory (e.g., calculations of contrast or a metric derivedtherefrom, waveforms formed by the light intensity measurements, etc.).The processor 500 may be configured to read from the memory. Forexample, the processor 500 may read raw input from the photodetector 200stored in the memory or partially processed input and perform furtheroperations on the data (e.g., calculation of a volumetric flow rate froma metric of contrast, calibration of a measurement, etc.). Data storedin the memory may be stored short-term or long-term. For example, theprocessor 500 may send and retrieve input data to and from the memorywhile simultaneously performing operations on input from thephotodetector 200 as the input is being generated and/or transmitted tothe processor 500. The processor 500 may store data, measurements,calculations, and the like, from previous measurements uses of theinterrogation device 400 or store data from another interrogation deviceto be used in the processing of subsequent input from the interrogationdevice 400 (e.g., for calibration, as described elsewhere herein). Thedata stored in the memory may be written to the memory by the processor500 or another processor operatively coupled to the memory. The storeddata may be generated from the interrogation device 400, generated byanother interrogation device 400, or generated by other means (e.g.,input by a user into a computer or input into an interrogation device400).

In some embodiments, the processor(s) and/or memory used in determiningparticle characteristics, including for example calibrating measurementsderived from photodetector 200 input, may be integrated into theinterrogation device 400. In some implementations, the interrogationdevice 400 may store measurements from previous interrogations. Forexample, measurements made on one or more calibration samples includinglight scattering particles with known particle characteristics, asdescribed elsewhere herein, may be stored locally within theinterrogation device 400 and used by the processor(s) for calibratingsubsequent measurements of samples with unknown particlecharacteristics. Similarly, data relating to components of theinterrogation device 400, such as estimates of sensor noise or lightsource coherence length, may be stored locally on the interrogationdevice 400 and used by the processor for calibrating measurements. Insome embodiments, the system may comprise a pre-calibrated devicewithout the need to interface with an external processor or memory. Thecalibration may be performed as part of the manufacturing process or maybe subsequently calibrated. The systems and methods disclosed herein canbe practiced according to any combination of processor(s) and memory.The processor and memory may be both integrated into the interrogationdevice 400 (i.e. internal) or both external to the interrogation device400. The memory may be internal and the processor external orvice-versa. In some implementations, the calibrations disclosed hereinmay be performed using both internal and external processors and/orusing internal and external memory.

The disclosed devices, systems, and methods employ an innovative conceptof reducing the susceptibility of speckle images to noise anddeleterious speckle pattern formulation effects by calibrating thesensor output. In some embodiments, the output may be calibrated byperforming measurements of samples comprising light scattering particleswith particle characteristics known a priori (e.g., known motion and/orlight intensity variance). Correlating future measurements from sampleswith unknown particle motion characteristics to these measurements fromcalibration samples in combination with data related to the a prioriknown particle characteristics, or to data derived from a combinedanalysis of the measurements and known particle characteristics (e.g. acalibration function or model), may be used to correct for unwantedsignals in the measurements and inform the samples of interest. Thecorrection in subsequent contrast measurements can be used to moreaccurately determine particle motion characteristics.

Particle motion characteristics may generally be derived from measuringcontrast in the disclosed systems. Panicle motion characteristics mayinclude volumetric flow rates of particles, diffusion coefficients (fromwhich particle size and viscosity may be derived), degrees oflaminarity/turbulence, hematocrit, blood perfusion in biologicalsamples, etc. Other particle characteristics may include, for example,optical particle characteristics, such as absorption spectrum,absorption coefficients, scattering coefficients, reduced scatteringcoefficients, scattering anisotropy, etc. A priori knowledge of particlemotion characteristics (e.g., flow rate) in a calibration sample may beused to correct measurements so that the determined particle motioncharacteristics of the calibration sample would match the true valuesknown a priori. Non-motion particle characteristics (e.g., opticalparticle characteristics) may affect the measurement of contrast andultimate determination of particle motion characteristics. For instance,high levels of absorption by light scattering particles within a samplemay affect (e.g., increase) contrast. A priori knowledge of thesecharacteristics may also be used to correct contrast measurements. Forexample, measurements of calibration samples with unknown flow rates butknown optical particle characteristics, such as absorption coefficient,may be used to adjust for that optical particle characteristic in futuresamples of interest. For instance, a calibration sample withsubstantially 0% absorption could be measured at an unknown flow rateand the absorption coefficient increased a known amount, such as byadding an absorbing dye to the sample while at the same flow rate. Themeasured change in contrast as a result of absorption coefficient couldbe stored to memory and used to correct future empirical measurements ofcontrast for a sample of interest with unknown particle motioncharacteristics but a known (e.g., measurable) absorption coefficient.

In some embodiments, the output may be calibrated by determining an apriori estimate for the amount of unwanted signal affecting totalmeasured contrast, which may or may not be based on prior LSImeasurements, and correcting empirical measurements in samples ofinterest by accounting for the estimated unwanted effect on contrast. Insome implementations, the correction may comprise a simple mathematicaloperation, such as a subtraction/addition or multiplication/division.The correction may, in a non-limiting example, take the form of simpledivision or subtraction of the signal derived from a relativelymotionless element or object. In one embodiment, the amount of darkcurrent in the sensor pixels could be estimated a priori based on amanufacturer specification. The estimate of the dark current could thenbe referenced to predict the undesired effect on pixel intensityvariance and/or mean intensity, and finally subtracted from theempirical contrast calculation to estimate the noise-free contrast. Insome embodiments, contrast measured from a static calibration samplearising from undesired signals may be subtracted from futuremeasurements. For example, the contrast for a static object exhibitingno flow, such as a piece of paper, may be assumed to have a theoreticalcontrast of 1 when the actual measured contrast is less than 1. Anydeviation from the expected result may be assumed to arise fromimperfections in the system components (e.g., finite laser coherence orpolarization, pixel size, sensor non-linearity, system optics, etc.).All future measurements could be divided by the calibration value (e.g.,0.8) to correct for the error. The correction may, in anothernon-limiting example, take the form of creating a correctivelookup-table or analytical calibration function.

Disclosed herein are novel methods, systems, and devices for thecalibration of speckle contrast flowmetry measurements using previouslyrecorded data from samples at a known volumetric flow or known orexpected contrast. Broadly, the disclosure relates to an innovativemethod to calibrate a dynamic light scattering measurement, and inparticular the speckle contrast analysis method. The LSI devicesdisclosed herein are configured to measure the optical contrast detectedby the one or more pixels of the photodetector and may be referred to aslaser speckle contrast analysis devices. Advantageously, the imagesdetected by the photodetector 200 of the present disclosure can beunfocused. The rate of motion (e.g., flow rate) can be determined from aglobal average of the detected speckle contrast rather than by mappingthe detected speckle pattern to focused light scattering particles.Configuring the photodetector 200 to obtain focused images can beexpensive and spatially constraining. Photodetectors 200 configured toaccept unfocused light may advantageously be smaller and may be moresuitable to be worn by a user. As such, the photodetector 200 may beconfigured to accept unfocused (i.e. non-convergent) light rays. Forexample, the photodetector 200 may be configured to accept raw unalteredlight paths that have not been altered by optical elements, such as alens, which modify the path or direction of the impinging light.

The method of speckle contrast imaging is commonly used to image vesselsand vascularized tissues within the field of biomedical engineering andmedicine [7]. The method takes advantage of the interference patternformed when coherent laser light scatters randomly in a sample media.The so-called speckle pattern is formed onto an image sensor. If thescattering objects are in motion, the speckle pattern will fluctuateduring the exposure time of the image sensor, which will cause ablurring of the pattern. For a given camera exposure, fasterfluctuations induce more blurring. One measure of the “blur” in aspeckle image is commonly referred to as the speckle contrast, and isconventionally defined as:K=σ/<I>  [1]where σ is the standard deviation and <I> is the mean of N pixelintensities (for a silicon-based image sensor, the pixel intensity isproportional to the voltage output from the detector element). Othermeasures of contrast can be used as well, with contrast being definedgenerally as any measure of disparity, difference, or distinctionbetween values of multiple pixel elements of the photodetector 200,and/or the evolution of a single pixel element over time. Non-limitingexamples include statistical properties of the spatial or temporalcontrast, such as the speckle flow index (defined as k₀/K² where K isthe speckle contrast as described herein and k₀ is a constant), standarddeviation from mean or median, difference metrics such as mean percentdifference (e.g., between pixels of the photodetector 200),potential-well fill time difference, gradient between pixels, metrics ofcomparisons between subregions such as subtraction, the magnitude offluctuation in the pixel intensities over time, reduction of the pixelsto local binary patterns or local ternary patterns, etc. Anautocorrelation performed on the signal generated by a single pixel overa period of time may quantify the temporal decorrelation in detectedlight intensity as a result of the motion of the moving light scatteringparticles.

As a non-limiting example of relating a metric of contrast to the flowrate of moving particles, the spatial speckle contrast can be related tothe autocorrelation time of the speckle image, which can then be relatedto the mean square displacement (e.g. flow speed or diffusion) of themoving scattering objects [6]. In general, a relatively high contrastspeckle pattern will produce higher values of K and a more blurrypattern will produce lower values of K. The rate of movement (e.g.,flow) within a sample can then be related to the contrast, which can becomputed either through analytic or empirical means. It should also benoted that temporal calculations of K, where contrast is derived from asingle optical detection element over time can be used interchangeablywith spatial computations of contrast. Temporal calculations of K dependon the arithmetic comparison of different intensity values within asingle optical element over a period of time. In this case, multiplevalues for a single optical element collected over a sequence of timeare compared to one another, as opposed to the comparison of values ofan optical detection element to that of its surrounding neighbors at thesame moment in time. While temporal calculations of K involve thecomparison of a single optical element to itself, by comparing differentvalues detected over time, the ultimate calculation of K can and ofteninvolves multiple optical detection elements. Additionally, combinationsof spatial and temporal calculations of contrast may also be usedwithout a loss of generality. In some embodiments, the rate of movementmay be determined as the speed, or average speed (e.g., m/s), of themoving light scatterers within a sample. The flow rate may be a measureof the volume of fluid (e.g., blood) transported per unit of time (i.e.volumetric flow) and may be represented in any suitable units (e.g.,cm³/s). In some embodiments, the flow rate may be determined as ameasure of volumetric flux (e.g., m³·s⁻¹·m⁻²) through, for example, ablood vessel or blood vessels.

The present disclosure relates to novel devices, systems, and methodsfor calibrating and/or correcting the speckle contrast, in a manner thataccounts for detector noise and/or other non-flow factors that may causeundesired errors in a measurement. In some embodiments, the calibrationstep involves measurement of known or expected contrast. The measurementof known or expected contrast may be used to correct subsequentmeasurements of unknown contrast, prior to determining unknown particlecharacteristics in a sample of interest. For example, the expectedcontrast during illumination under incoherent light is 0. If thecontrast measurement of a speckle flowmetry system is not 0 in theseconditions, the contrast may be corrected to achieve the expected resultof 0. FIG. 4 illustrates an example of a measured speckle flow metric,the speckle contrast K as defined by eq. 1, averaged across thephotodetector 200, illuminated by incoherent light. The measuredcontrast is expected to be 0 at all intensities of light, yet isnon-zero at all measured intensities. The measured contrast drasticallyincreases as the intensity of light is reduced. The nonzero specklecontrast can be due to camera noise effects, which vary as a function ofintensity. To correct for this error in the measure of contrast, thevalues plotted here for each intensity can be subtracted from futurecontrast measurements as a process of calibration to account for cameranoise.

In a non-limiting case, the detector noise and other non-flow elementsmay be assumed to be an additive term to the variance, σ², betweenpixels, and is described as:σ² _(measured)=σ² _(true)+σ² _(noise).  [2]Under illumination of incoherent light, as described in the exampleabove, σ_(true) is presumed to be 0. Thus, σ_(noise) can be solved foralgebraically. The term σ_(noise) may be assumed to be constant noisefrom factors such as camera current, shot noise, ambient light, etc.Thus, σ_(noise) may be subtracted from future measurements to eliminateeffects from noise parameters and determine σ_(true). The value ofσ_(true) can then be used further to determine a metric of calibratedcontrast by using, for example, the conventional contrast in eq. 1. Someembodiments can comprise other ways to determine or estimate σ_(noise).These may be, but are not limited to, a priori estimates from thespecifications of the sensor pixels or coherent source, through expectedstochastic process statistics, through measurement of background lightby a different sensor, or by assuming equivalent performance as othersystems (e.g., interrogation devices) already measured.

In some embodiments, the calibration step involves the measurement ofcontrast for a sample with a known particle characteristic (e.g., flowrate). In one non-limiting example, a sample of light scattering fluid(a fluid comprising light scattering particles) may be pumped at a knownvolumetric flow rate through a tube, channel, or other container. Aportion or the entirety of the tube, channel, or container may betransparent to optimize interrogation of the fluid with light. Thesample may then be illuminated by coherent light, and the contrastvalues of the detection system recorded for varying rates of flow.Unknown particle motion characteristics from new samples of interest maythen be determined by comparing the measured contrast to that measuredfor the sample with known flow.

A calibration function can be determined using contrast measurementsderived from known volumetric flow rates. One may assume the contrastcan be related to the volumetric flow through an unknown function:Flow=f(K).  [3]The speckle contrast, K, or any other suitable measure of contrast maybe employed by the function. The term f(K) may be assumed to be anunknown function, which may be approximated through simulation, analyticmodeling work, or left unknown. Through measurements of known flow, f(K)may be determined empirically. In a non-limiting case, the function maybe assumed to be continuous, and a table of Flow vs. K pairs may becreated, wherein future measurements of K may be interpolated orextrapolated between known pairs. For example, a data-set may be storedto memory comprising a look-up table. The look-up table can includepairs of measurements and known particle characteristics. For instance,each pair may include a measure of contrast generated by interrogating asample with known particle characteristics and the associated knownparticle characteristic (e.g., value of the flow rate or absorptioncoefficient). The look-up table may, for example, include, a range ofknown flow rates of fluid comprising light scattering particles pumpedthrough a calibration sample, the flow rates being selected across acontinuous range of flow, and the respective measures of contrastderived from the photodetector 200 input as measured from thecalibration sample for each known flow rate. The processor may then usethe look-up table to interpolate the unknown particle characteristic(e.g., flow rate) of a measured sample of interest by comparing theempirically measured contrast to the stored measures of contrast in thelook-up table. The processor may assume the true particle characteristicof the sample of interest lies between values of the stored particlecharacteristics corresponding to the measures of contrast immediatelygreater than and immediately less than the measured contrast of theunknown sample. In some implementations, the processor may assume alinear relationship between the measure of contrast and the particlecharacteristic between immediately adjacent stored data pairs.

In a second non-limiting case, f(K) may be determined through neuralnetworks, where future measurements of K may be fed into the forwardnetwork, which then outputs a best approximation of the Flow metric.Calibration measurements of K for known flow rates may be used to trainthe neural network. Using larger numbers of calibration measurements mayresult in a more accurate neural network.

In a third non-limiting case, f(K) may be approximated throughsimulation or analytic modeling work, and any unknown parameters withinthe model may be estimated or solved for by comparing the Flow vs. Kpairs. Simulations may rely on random number generators and assumedprobability distributions to approximate the contrast for particles ofknown flow rates. For example, a Monte Carlo simulation can be usedsimulate the path of many photons, including scattering angles andlength between scattering events, to statistically calculate the measureof contrast across multiple flow rates. Interpolation may be used toaccurately determine unknown flow rates from measured values ofcontrast. Analytical approximations may use some approximation ofscattered photon properties (e.g., a diffusion equations) to determine acontinuous closed form solution which can be evaluated for any measureof contrast. For example, an assumed particle velocity distribution(e.g., Lorenztian) may be used to estimate an autocorrelation functionof the remitted light, which could be integrated over time toapproximate contrast as a continuous function of velocity. Theanalytical model can include a variable term (e.g., a scalar multiple,exponent, additive term, etc.) to account for deviation from thepredicted solution. The variable term could be determined for a givensystem by using an optimizer to fit the analytical model, keeping thevariable term as a free term, to a set of empirically determined datafrom a calibration sample. The resolved variable term could be used tomore accurately determine particle motion characteristics from futurecontrast measurements using the analytical model.

In a fourth non-limiting case, the function f(K) may be estimated bycommon functions, such as polynomial series, exponential function,geometric function (e.g., sine, cosine, tangent), Fourier series, Taylorseries, statistical distribution (e.g., Gaussian), or other function,wherein future values of K may be inserted into the expression to yielda value of Flow. The scope of the present disclosure includes all othermeans for determining a relationship between f(K) and flow usingpreviously determined measurements at known flow values.

FIG. 5 illustrates an example of a calculated speckle flow metric, theflow index (defined elsewhere herein), averaged across the photodetector200, as measured for a calibration sample subjected to known flow ratesof light scattering particles. The flow index is approximately linearwith flow. The values plotted can be used to generate a look-up table ofvalues, where future values of flow index or K in a sample of unknownconditions can be interpolated into, as described elsewhere herein.Alternatively, the equation of the fitted line may be solved and futurevalues of flow index or K converted to true measures of volumetric flowusing the linear approximation, as described elsewhere herein.

A calibration can also be performed using physiological measurementsunder known or expected conditions. For example, during an occlusion ofthe extremities, there is a cessation or significant reduction of bloodflow to the hands and/or feet. An occlusion can be carried out using adevice such as, but not limited to, a blood-pressure cuff often placedover the ankle, to produce cessation of blood flow to the feet, and overthe bicep to produce cessation of blood flow to the hands. After bloodflow is stopped to the hands and or feet, the measured value is expectedto represent a state of no flow and can be offset as such. This form ofcalibration can allow for customization due to subject-to-subjectvariability, and can be carried out independently or used in conjunctionwith the other aforementioned calibration methods. A physiologicalmethod of calibration can also aid in calibrating for differencesbetween a subject's own hands and feet, for instance. Furthermore, whilethe examples presented above are illustrated with hands and feet, thismethodology can be applied to any measurement of blood flow within thevascularized tissue.

The disclosed systems and methods may produce a more reliable devicewith applications in healthcare and wearable technology. For example,the system could provide more accurate measurements of flow, or providea larger pulsatile amplitude for detecting the cardiac waveform. Asystem could be integrated into a wearable wrist monitor, to performblood flow monitoring or heart rate monitoring. The blood flow and heartrate monitoring could be improved using the calibration techniquedescribed above. In a second non-limiting example, a system couldminiaturized and placed on a medical device intended to monitor vascularhealth, where the vascular flow can be made more accurate throughcalibration. In this example, the medical device could be affixed totissue of interest to clinicians and the disclosed system and methodcould be used to measure the flow of red blood cells within this tissue.Specifically, the medical device could, for example, be affixed to apatient's foot so that blood flow could be quantified in this tissueusing the disclosed system and method. In such tissue (and others),blood circulation is required to deliver oxygen and remove cellularwaste products. As such, a minimal amount of blood flow is required tosustain continued tissue viability such that nutrient delivery isadequate to meet metabolic tissue demands. The disclosed system andmethod could thus be used to measure blood flow (circulation) in thetissue for the purpose of determining whether the measured quantity isconsistent with continued tissue viability, and as such, be used toassess the degree of blood circulation adequacy. The processor may beprogrammed to compare the measured blood flow (circulation) to apredetermined value and determine whether the blood circulation isadequate.

WORKING EXAMPLE

FIG. 6 illustrates an example of data output from an interrogationdevice, such as illustrated in FIGS. 2A and 2B, and operated accordingto the methods and systems described herein. The measured waveformscorrelate to the pulsatile blood flow originating from the cardiaccycle. The pulsatility reflects the changes in the volumetric flow rateas the subject's heart pumps blood through the interrogated vasculature.The periodicity of the flow arises from the cardiac cycle and can beused to determine heart rate by determining the period betweensuccessive waveform features (such as systolic contraction peaks). Theoutput from the photodetector 200 is shown before accounting for noiseand non-flow elements and after non-flow elements are accounted forthrough calibration. The calibration in this example was performedutilizing previously recorded contrast data on a calibration samplesubject to static flow and interrogated under incoherent lightconditions. The noise measured during calibration was subtracted fromthe present photodetector measurements recorded over time. As shown inFIG. 6 , the calibration effectively reduced the measured non-pulsatilecontrast elements, essentially amplifying the true flow signal.

While the present invention has been described in terms of particularembodiments and applications, in both summarized and detailed forms, itis not intended that these descriptions in any way limit its scope toany such embodiments and applications, and it will be understood thatmany substitutions, changes and variations in the described embodiments,applications and details of the method and system illustrated herein andof their operation can be made by those skilled in the art withoutdeparting from the spirit of this invention.

REFERENCES (INCORPORATED HEREIN BY REFERENCE THERETO)

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What is claimed is:
 1. A system for determining unknown particle motioncharacteristics in a sample of interest using a calibrated contrastmeasurement from a laser speckle imaging device, the system comprising:a laser speckle imaging device configured for contrast analysiscomprising: a light source configured to emit light such that the lightscatters within a sample; and a photo-sensitive detector comprising oneor more light-sensitive pixel elements configured to receive at leastsome of the light; a computer-readable memory storing calibration data,the calibration data comprising: an a priori estimate of the effect oncontrast arising from signals unrelated to particle motioncharacteristics of the light scattering particles in the sample ofinterest; and a processor operably coupled to the detector and to thecomputer-readable memory, the processor being programmed to: derive anempirical measure of the total contrast in light detected by the one ormore pixel elements in time and/or space that has scattered from thesample of interest comprising light scattering particles with unknownparticle motion characteristics; calibrate the empirical measure oftotal contrast by using the a priori estimate to correct for contrastelements that are unrelated to particle motion characteristics of thelight scattering particles of the sample of interest; and determine theunknown particle motion characteristics of the sample of interest fromthe calibrated empirical measure of total contrast.
 2. The system ofclaim 1, wherein the a priori estimate is based on at least onepreviously recorded measurement.
 3. The system of claim 2, wherein theat least one previously recorded measurement was taken using incoherentlight.
 4. The system of claim 2, wherein the at least one previouslyrecorded measurement was recorded using the same laser speckle imagingdevice used to detect the light scattered from the sample of interest inderiving the empirical measure of the total contrast.
 5. The system ofclaim 1, wherein the a priori estimate is based at least in part on thenoise characteristics of the detector.
 6. The system of claim 1, whereinthe a priori estimate is based at least in part on ambient or backgroundlight.
 7. The system of claim 1, wherein the a priori estimate is basedat least in part on light intensity variation not due to interference.8. The system of claim 1, wherein the light scattering particles of thesample of interest are blood cells and the unknown particlecharacteristics comprise a measure of the flow rate of the blood cells.9. The system of claims 1, wherein the empirical measure of totalcontrast comprises a measure of pixel variance and the a priori estimatecomprises an estimate of pixel variance, and wherein correcting theempirical measure comprises subtracting or ratioing the a prioriestimate of variance from the empirical measure of variance.
 10. Thesystem of claim 1, wherein the laser speckle imaging device, thecomputer-readable memory, and the processor are housed within a singledevice.
 11. The system of claim 10, wherein the single device isconfigured to be worn by a user to measure a sample of interest withinthe user.
 12. The system of any of claim 1, wherein the laser speckleimaging device is configured to measure pulsatile blood flow derivingfrom the cardiac cycle.
 13. A method for determining unknown particlemotion characteristics in a sample of interest using a calibratedcontrast measurement from a laser speckle imaging device, the methodcomprising: employing a laser speckle imaging device configured forcontrast analysis to obtain a measurement of light scattered from asample of interest comprising light scattering particles with unknownparticle motion characteristics, the laser speckle imaging devicecomprising: a light source configured to emit light such that the lightscatters within a sample; and a photo-sensitive detector comprising oneor more light-sensitive pixel elements configured to receive at leastsome of the scattered light; accessing from computer-readable memory ana priori estimate of the effect on contrast arising from signalsunrelated to particle motion characteristics of the light scatteringparticles of the sample of interest; deriving an empirical measure ofthe total contrast in light detected by the one or more pixel elementsin time and/or space from the measurement of light; calibrating theempirical measure of total contrast by using the a priori estimate tocorrect for contrast elements that are unrelated to particle motioncharacteristics of the light scattering particles of the sample ofinterest; and determining the unknown particle motion characteristics ofthe sample of interest from the calibrated empirical measure of totalcontrast.
 14. The method of claim 13, further comprising employing thelaser speckle imaging device to obtain the a priori estimate.
 15. Themethod of claim 14, wherein employing the laser speckle imaging deviceto obtain the a priori estimate comprises pumping fluid comprising lightscattering particles with particle characteristics known a priori at aknown flow rate and measuring light scattered from the light scatteringparticles with particle characteristics known a priori.
 16. The methodof claim 15, wherein pumping the fluid at a known flow rate comprisespumping the fluid at two or more different known flow rates.
 17. Themethod of claim 14, wherein employing the laser speckle imaging deviceto obtain the a priori estimate comprises occluding blood flow within anextremity of a living subject to reduce or cause a cessation of bloodflow and measuring light scattered from the occluded extremity of thesubject.
 18. The method of claim 17, wherein occluding blood flowcomprises applying a blood-pressure cuff to the ankle, legs, or arms ofthe subject.
 19. The method of claim 13, further comprising illuminatinga calibration sample with incoherent light to obtain the a prioriestimate.
 20. The method of claim 13, further comprising: employing thelaser speckle imaging device to obtain a subsequent measurement of lightscattered from the same or a different sample of interest comprisinglight scattering particles with unknown particle motion characteristics;accessing the a priori estimate from the computer-readable memory;deriving a subsequent empirical measure of the total contrast in lightdetected by the one or more pixel elements in time and/or space from thesubsequent measurement of light; calibrating the subsequent empiricalmeasure of total contrast by using the a priori estimate to correct forcontrast elements that are unrelated to particle motion characteristicsof the light scattering particles; and determining the unknown particlemotion characteristics from the calibrated subsequent empirical measureof total contrast.
 21. The method of claim 13, wherein the lightscattering particles of the sample of interest are blood cells anddetermining the unknown particle characteristics comprises determiningthe flow rate of the blood cells.